1. Technical Field
The present invention relates generally to nucleic acid immunization. In particular, the invention relates to an infection/transfection system for nucleic acid immunization which stimulates a cell-mediated immune response in a vertebrate subject.
2. Background of the Invention
Cytotoxic T-lymphocytes (CTLs) play an important role in cell-mediated immune defense against intracellular pathogens such as viruses and tumor-specific antigens produced by malignant cells. CTLs mediate cytotoxicity of virally infected cells by recognizing viral determinants in conjunction with class I MHC molecules displayed by the infected cells. Cytoplasmic expression of proteins is a prerequisite for class I MHC processing and presentation of antigenic peptides to CTLS. However, immunization with killed or attenuated viruses often fails to produce the CTLs necessary to curb intracellular infection. Furthermore, conventional vaccination techniques against viruses displaying marked genetic heterogeneity and/or rapid mutation rates that facilitate selection of immune escape variants, such as the human immunodeficiency virus (HIV) or influenza, are problematic.
Recently, a technique for the direct injection of DNA and mRNA into mammalian tissue, has been described. See, e.g., International Publication No. WO 90/11092 (published Oct. 4, 1990). The method, termed xe2x80x9cnucleic acid immunizationxe2x80x9d herein, has been shown to elicit both humoral and cell-mediated immune responses. For example, sera from mice immunized with a human immunodeficiency virus type 1 (HIV-1) DNA construct encoding the envelope glycoprotein, gp160, were shown to react with recombinant gp160 in immunoassays and lymphocytes from the injected mice were shown to proliferate in response to recombinant gp120. Wang et al. Proc. Natl. Acad. Sci. USA (1993) 90:4156-4160. Similarly, mice immunized with a plasmid containing a genomic copy of the human growth hormone (hGH) gene, demonstrated an antibody-based immune response. Tang et al. Nature (1992) 356:152-154. Intramuscular injection of DNA encoding influenza nucleoprotein driven by a mammalian promoter has been shown to elicit a CD8+ CTL response that can protect mice against subsequent lethal challenge with virus. Ulmer et al. Science (1993) 259:1745-1749. Immunohistochemical studies of the injection site revealed that the DNA was taken up by myeloblasts, and cytoplasmic production of viral protein could be demonstrated for at least 6 months.
However, safety issues limit the usefulness of nucleic acid immunization. In particular, there is the potential for integration of unwanted, foreign DNA into the recipient""s genome and the possibility of long-term expression of a protein that is potentially harmful to the host. A possible solution to the latter problem is by employing a system that assures the transient, controlled expression of the administered sequence in the host cell.
A vaccinia based infection/transfection system has been developed (Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126) which provides for the transient expression of a protein sequence in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with a DNA of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747. The system has been used to study protein processing and subcellular localization, as well as for the identification of proteins encoded by various viral genomes. See, e.g., Tomei et al. J. Virol. (1993) 67:4017-4026; Selby et al. J. Gen. Virol. (1993) 74:1103-1113. The system has recently been shown to be useful for the identification of CTL epitopes from influenza gene products. Eisenlohr et al. J. Immunol. Meth. (1992) 154:131-138.
However, none of the above-described art provides an infection/transfection method of nucleic acid immunization which allows the transient, cytoplasmic expression of a selected antigen in order to elicit a class I MHC restricted CTL response.
The present invention is based on the discovery of a system for nucleic acid immunization which is effective in eliciting a cell-mediated immune response to a particular antigen. The invention takes advantage of an infection/transfection system which provides for the controlled, transient cytoplasmic expression of a given antigen and which elicits the production of class I MHC restricted CTLS.
Accordingly, in one embodiment, the invention is directed to a method of nucleic acid immunization which results in a cell-mediated immunological response to a selected antigen. The method comprises immunizing a vertebrate subject by:
(a) introducing a source of bacteriophage T7 RNA polymerase into a cell of the subject; and
(b) transfecting the cell with a recombinant vector comprising a coding sequence which encodes the selected antigen operably linked to the bacteriophage T7 promoter, wherein the transfecting is done under conditions whereby the coding sequence is transcribed and translated in the cell,
thereby resulting in the production of a cell-mediated immunological response in said vertebrate subject.
In particularly preferred embodiments, the T7 polymerase is introduced using a recombinant vaccinia virus, the recombinant vector is liposome-encapsulated and the selected antigen is a viral antigen such as a human immunodeficiency virus (HIV) or a hepatitis C virus (HCV) envelope glycoprotein.
In another embodiment, the invention is directed to a vaccine composition comprising a recombinant vector which includes a coding sequence for a selected antigen operably linked to the bacteriophage T7 promoter, in admixture with a pharmaceutically acceptable excipient.
In yet another embodiment, the invention is directed to a method of making a vaccine composition which comprises admixing a recombinant vector which includes a coding sequence for a selected antigen operably linked to the bacteriophage T7 promoter, with a pharmaceutically acceptable excipient.
These and other embodiments of the subject invention will readily occur to those of skill in the art in light of the disclosure herein.